CN112939905A - Compound with aggregation-induced emission property and preparation method and application thereof - Google Patents

Abstract

The invention relates to a compound with aggregation-induced emission property, a preparation method and application thereof, wherein the compound has a molecular structural formula

Wherein R is¹、R²Each independently selected from H,

One kind of (1). The invention provides a compound with aggregation-induced emission propertiesThe compound has longer absorption and emission wavelength, the emission wavelength can reach a near infrared region, and the compound has high type I ROS generation efficiency. The compound with aggregation-induced emission properties provided by the invention can be used for targeted tumor cell nucleus imaging after being wrapped into nanoparticles by a cell nucleus targeting carrier. Can be used for the targeted photodynamic therapy of tumor cell nucleus.

Description

Compound with aggregation-induced emission property and preparation method and application thereof

Technical Field

The invention relates to the technical field of multifunctional organic small molecule synthesis, in particular to a compound with aggregation-induced emission properties, and a preparation method and application thereof.

Background

Photodynamic Therapy (PDT), a novel and effective cancer treatment modality, has attracted attention because of its unique advantages of high spatiotemporal accuracy, controllability, non-invasiveness, low toxic and side effects, and the like. Most current organic photosensitizers are based on the generation of type II reactive oxygen species (such as singlet oxygen,¹O₂) Mainly comprises the following steps of (1) taking the main part,¹O₂the generation of the tumor has high oxygen dependence, and the hypoxic property of the tumor part is not favorable¹O₂The PDT effect is mainly exerted, and the oxygen loss in the PDT process further reduces the curative effect.

Type I photosensitizers can utilize superoxide dismutase mediated disproportionation reactions to produce abundant free radical reactive oxygen species (type I reactive oxygen species) within cells. In type I PDT, photosensitizers generate superoxide anions by electron transfer with substrates (e.g. reducing coenzymes, amino acids, vitamins and nitrogenous bases, etc.), oxygen ((^·O₂ ^-) Peroxide (H)₂O₂) And a hydroxyl radical (C^·OH). Wherein the content of the first and second substances,^·OH is the most biologically active one of these reactive oxygen species and can cause irreversible destruction of almost all biomolecules. In addition, the generation of free radical active oxygen species is accompanied with the cyclic utilization of oxygen, so that the utilization rate of oxygen in cells can be greatly improved, and the problem of tumor hypoxia is effectively solved.

At present, I-type photosensitizers based on small organic molecules are not rare, and in few reports, the small organic molecules are influenced by aggregation-induced quenching (ACQ) effect, so that the problems of reduced fluorescence intensity and reduced generation efficiency of active oxygen are easy to occur in an aggregation state.

Accordingly, the prior art is yet to be improved and developed.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a compound having aggregation-induced emission properties, a preparation method and applications thereof, and aims to solve the problems that the conventional photosensitizer is prone to decrease in fluorescence intensity and decrease in active oxygen generation efficiency in an aggregated state.

The technical scheme adopted by the invention for solving the technical problems is as follows:

in a first aspect, a compound having aggregation-induced emission properties has the following molecular formula

Wherein R is¹、R²Each independently selected from H,

One kind of (1).

In a second aspect, a method of preparing a compound having aggregation-induced emission properties as described above, wherein when R is¹、R²When both are H, the method comprises the steps of:

providing a compound I;

dissolving the compound I and malononitrile into ethanol, and heating and refluxing to obtain the R¹、R²A compound each independently selected from H;

wherein the molecular structural formula of the compound I is

Optionally, the compound preparation method, wherein the compound I preparation method comprises the steps of:

adding 4-bromotriphenylamine, 5-formaldehyde furan-2-boric acid, [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride and potassium carbonate into a reaction vessel;

and adding the mixed solution into the reaction vessel, and heating the reaction vessel under an inert atmosphere to obtain the compound I.

Alternatively, the compound production method, wherein the mixed solvent contains methanol and toluene.

Alternatively, the compound preparation method, wherein, when R is¹Is H, R²Is composed of

The method comprises the following steps:

providing a compound IV;

dissolving the compound IV and malononitrile in dry ethanol, and heating and refluxing to obtain R¹Is H, R²Is composed of

A compound of (1);

the molecular structural formula of the compound IV is

Optionally, the compound preparation method, wherein the preparation of the compound IV comprises the following steps:

providing a compound III;

adding the compound III, 5-formaldehyde furan-2-boric acid, [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride and potassium carbonate into a reaction vessel;

adding a mixed solvent containing methanol and toluene into the reaction container, and heating the reaction container under an inert atmosphere to obtain a compound III;

the molecular structural formula of the III is

Optionally, the compound preparation method, wherein the preparation of the compound III comprises the following steps:

providing a compound II;

adding the compound II, p-bromoiodobenzene, 1, 10-phenanthroline, cuprous iodide and potassium hydroxide into a reaction vessel;

adding toluene under the protection of inert gas, and heating the reaction container to obtain the compound III;

the molecular structural formula of the compound II is

Optionally, the compound preparation method, wherein the preparation of the compound II comprises the following steps:

dissolving 1- (4-bromophenyl) -1, 2, 2-triphenylethylene, aniline, tri-tert-butylphosphine, tris (dibenzylideneacetone) dipalladium and sodium tert-butoxide in toluene;

and (3) carrying out reaction under the protection of inert gas and heating condition to obtain the compound II.

Optionally, the compound preparation method, wherein the heating conditions are heating temperature of 110-.

In a third aspect, use of a compound having aggregation-induced emission properties for nuclear-targeted imaging or for nuclear-targeted photodynamic therapy.

Has the advantages that: the compound with aggregation-induced emission property provided by the invention has longer absorption and emission wavelength which can reach a near infrared region. Has high type I ROS generation efficiency.

Drawings

FIG. 1 is a normalized ultraviolet absorption spectrum of TFMN and TTFMN in acetonitrile solution;

FIG. 2 is a graph showing the fluorescence emission spectrum of TTFMN (10. mu.M) in an acetonitrile/water (v/v) mixed solvent, as the water content increases, lambda_ex＝490nm；

FIG. 3 is a graph showing the fluorescence enhancement factor of TFMN and TTFMN (10. mu.M) in a mixed solvent of acetonitrile/water (v/v) with increasing water content, and the inset is an enlarged graph of the TFMN curve, lambda_ex＝490nm；

Fig. 4 shows fluorescence emission spectra of TFMN and TTFMN in solid state.

FIG. 5 shows the white light (22.1mW cm)^-2) Extended irradiation time, fluorescence of mixed TFMN and TTFMN (2. mu.M) with Total ROS indicator DCFHThe light intensity enhancement multiple;

FIG. 6 shows the white light (22.1mW cm)^-2) Extended exposure time, TFMN and TTFMN (2 μ M)^·The fluorescence intensity of the OH indicator HPF mixed solution is enhanced by multiple times;

FIG. 7 shows white light (200mW cm) with DMPO as capture agent in the presence of TFMN and TTFMN (1mM), respectively^-2) ESR signal before and after (1 minute);

FIG. 8 shows the white light (22.1mW cm)^-2) Extended exposure time, TFMN and TTFMN (2 μ M)^·O₂ ^-The fluorescence intensity of the indicator DHR123 mixed solution is enhanced by multiple;

FIG. 9 shows the white light (22.1mW cm)^-2) Extended exposure time, TFMN and TTFMN (2 μ M)¹O₂The fluorescence intensity enhancement multiple of the indicator SOSG mixed solution;

FIG. 10 shows white light (22.1mW cm) in the presence of TFMN, TTFMN, RB (2 μ M)^-2) The irradiation time is prolonged, and the irradiation time is prolonged,¹O₂the rate of decomposition of the indicator ABDA;

FIG. 11 is a particle size distribution and TEM image of TTFMN nanoparticles (TTFMN-NPs);

FIG. 12 shows TTFMN-NPs at H₂O, stability in PBS and PBS with 10% FBS;

FIG. 13 is the absorption and emission wavelengths of TTFMN-NPs;

FIG. 14 shows the fluorescence intensity of TTFMN in each treatment group, detected by flow cytometry after co-incubation of 4T1 cells with TTFMN-NNPs, TTFMN-NPs, TTFMN-NPs (pretreated in PBS at pH5.0 for 24 hours), and TTFMN-PNPs for 3 hours, respectively, at a nanoparticle concentration of 10. mu.g mL^-1；

FIG. 15 shows 4T1 cells and TTFMN-NPs (2. mu.g mL)^-1) After incubation for 1 hour and 6 hours respectively, CLSM images after co-staining with lysosome blue probes respectively;

FIG. 16 shows 4T1 cells and TTFMN-NPs (2. mu.g mL)^-1) After incubation for 1 hour, 6 hours and 12 hours respectively, CLSM images after co-staining with nuclear dyes respectively;

FIG. 174T 1 shows that the concentration of nanoparticles is 50 μm in the intracellular reactive oxygen species detection after different treatmentsg mL^-1；

FIG. 18 shows 4T1 cells incubated with different concentrations of TTFMN-NPs, dark/white (50mW cm)^-2) Cell viability after irradiation treatment;

FIG. 19 shows apoptosis assay of 4T1 cells after different treatments, and nanoparticle concentration is 50 μ g mL^-1；

FIG. 20 is a graph of fluorescence imaging of tumor-bearing mice at various time points after tail vein injection of TTFMN-NPs;

figure 21 is a plot of the growth of mouse tumors in different treatment groups (n-5, p < 0.001);

figure 22 shows the tumor weight (n-5, p <0.001) of mice in different treatment groups after treatment;

FIG. 23 is a photograph of H & E, TUNEL, Ki67 and CD31 staining of tumor sections from mice in different treatment groups after treatment was completed.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer and clearer, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The compound with aggregation-induced emission properties provided in the embodiments of the present invention has a molecular structural formula as follows:

specifically, wherein R¹、R²Each independently selected from H,

The compound with aggregation-induced emission property is an I-type aggregation-induced emission photosensitizer, has better hypoxia tolerance, can more effectively utilize limited oxygen in a tumor microenvironment in the PDT process, and improves the photodynamic treatment effect of tumors. Furthermore, compared to suctionThe AIE photosensitizer with the collection and emission wavelengths in the visible light region and the near-infrared luminous AIE photosensitizer have many advantages in tumor diagnosis and treatment, such as strong penetrating power and small light damage to organisms, can avoid interference of autofluorescence of the organisms on signal collection, and can greatly reduce light scattering and the like. The organic fluorescent micromolecules have aggregation-induced emission effect, and the molecules have stronger electron supply (D) -electron absorption (A) effect, so that the absorption and emission wavelength of the molecules is red-shifted, and the organic fluorescent micromolecules have deep red/near infrared fluorescence emission and stronger ROS generation capacity. ROS generated by the organic fluorescent micromolecules are mainly I-type^·OH, provides a prerequisite for type I photodynamic therapy.

Based on the same inventive concept, the invention also provides a preparation method of the compound with the aggregation-induced emission property, such as preparation R¹、R²The route for compounds each independently selected from H (TFMN) is as follows:

condition a: pd (dppf) Cl₂Potassium carbonate, methanol/methanol mixed solvent, 75 ℃, 16 h; condition B: ethanol, reflux, 72 h.

(1) Synthesis of Compound 1

Weighing 4-bromotriphenylamine (324.2mg,1.0mmol), 5-formaldehyde furan-2-boronic acid (279.8mg,2.0mmol), [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (73.1mg,0.1mmol) and potassium carbonate (414.6mg,3.0mmol) in a 50mL three-necked flask, adding a mixed solution of methanol (5mL) and toluene (5mL), heating to 75 ℃ under the protection of nitrogen, reacting for 16h, cooling to room temperature after the reaction is finished, removing the solvent under reduced pressure, dissolving the residue with dichloromethane (100mL), then washing with water (3X 30mL), collecting the organic phase, drying with anhydrous sodium sulfate, concentrating, and separating by column chromatography to obtain 244.4mg of the target compound 1 with the yield of 72%.

(2) Synthesis of Compound TFMN

Compound 1(339.4mg,1.0mmol) and malononitrile (132.1mg,2.0mmol) were dissolved in dryHeating and refluxing in ethanol for 72h, concentrating, and separating by column chromatography to obtain 340.9mg of compound TFMN with 88.0% yield. Product nuclear magnetic and mass spectral characterization was as follows:¹H NMR(400MHz,Chloroform-d)δ7.68(d,J＝8.8Hz,2H),7.35-7.28(m,5H),7.25-7.2(m,1H),7.18-7.1(m,6H),7.10-7.04(m,2H),6.81(d,J＝3.9Hz,1H).¹³C NMR(100MHz,Chloroform-d)δ162.27,150.32,146.93,146.66,140.54,129.74,127.09,125.84,124.62,121.42,120.61,115.10,114.06,108.42,73.38.MALDI-MS:m/z calcd for C₂₆H₁₇N₃O 387.1372,found387.1364。

based on the same inventive concept, the invention also provides a preparation method of the compound with the aggregation-induced emission property, such as preparation R¹Selected from H, R²Is selected from

The route of the compound (TTFMN) of (a) is as follows:

condition a: pd (dppf) Cl₂Potassium carbonate, methanol/methanol mixed solvent, 75 ℃, 16 h; condition B: ethanol, refluxing for 72 hours; condition C is Pd₂(dba)₃Tri-tert-butylphosphine, sodium tert-butoxide, toluene solvent, 120 ℃ for 24 hours; condition D: cuprous iodide, 1, 10-phenanthroline, potassium hydroxide and a toluene solvent, wherein the temperature is 120 ℃ and the time is 48 hours.

(1) Synthesis of Compound 2

1- (4-bromophenyl) -1, 2, 2-triphenylethylene (205.0mg,0.5mmol), aniline (55.8mg,0.6mmol), tri-tert-butylphosphine (1.6mg,0.008mmol), tris (dibenzylideneacetone) dipalladium (6.4mg,0.007mmol) and sodium tert-butoxide (57.6mg,0.6mmol) were dissolved in dry toluene (10mL), heated to 120 ℃ under nitrogen, reacted for 24h.

(2) Synthesis of Compound 3

Weighing the compound 2(296.1mg, 0.7mmol), p-bromoiodobenzene (338.2mg,1.2mmol), 1, 10-phenanthroline (216.2mg,1.2mmol), cuprous iodide (228.5mg,1.2mmol) and potassium hydroxide (117.8mg,2.1mmol), adding the mixture into a 250mL double-neck bottle, adding toluene (50mL) to dissolve the mixture under the protection of nitrogen, heating the mixture to 120 ℃, reacting the mixture for 48h, cooling the mixture to room temperature after the reaction is finished, and removing the solvent by rotary evaporation under reduced pressure. The residue was dissolved in dichloromethane (100mL), washed with water (3 × 30mL), dried and subjected to column chromatography [ V (n-hexane): V (dichloromethane) ═ 50: 1] to give 143.8mg of compound 3 with a yield of 35.1%.

(3) Synthesis of Compound 4

Weighing the compound 3(374mg,0.65mmol), 5-formaldehyde furan-2-boronic acid (117.6mg,0.84mmol), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (47.3mg,0.06mmol) and potassium carbonate (446.7mg,3.2mmol) in a 50mL three-necked flask, adding a mixed solution of methanol (10mL) and toluene (10mL), heating to 75 ℃ under the protection of nitrogen, refluxing for 16h, after the reaction is finished, cooling to room temperature, removing the solvent under reduced pressure, dissolving the residue with dichloromethane (100mL), then washing with water (3X 50mL), collecting the organic phase, drying with anhydrous sodium sulfate, concentrating, and separating by silica gel column chromatography to obtain 210mg of the compound 4 with the yield of 54.7%.

(4) Synthesis of Compound TTFMN

Compound 4(140.1mg,0.236mmol) and malononitrile (62.4mg,0.943mmol) were dissolved in dry ethanol, heated under reflux for 72h, concentrated and isolated by column chromatography to give 130.1mg of compound TFMN in 85.9% yield.¹H NMR(400MHz,Chloroform-d)δ7.67(d,J＝8.8Hz,2H),7.27-7.33(m,4H),7.0-7.16(m,20H),6.94(d,J＝8.4Hz,2H),6.85(d,J＝8.8Hz,2H),6.8(d,J＝4.0Hz,1H).¹³C NMR(100MHz,Chloroform-d)δ162.08,149.91,146.76,146.28,144.56,143.81,143.50,143.26,141.12,140.35,139.90,132.46,131.33,131.30,131.28,129.48,127.71,127.62,127.60,126.86,126.53,126.43,125.56,124.46,124.38,121.42,120.47,114.96,113.93,108.27,73.15.ESI-HRMS:calcd.for C₄₆H₃₁N₃O[M]⁺:641.2467,found:641.2463。

As shown in fig. 1, the absorption peak of TFMN is 482 nm. Due to the introduction of TPE, TTFMN has stronger conjugation effect, and the absorption peak of TTFMN is red-shifted to 490 nm. The TFMN fluorescence intensity maximum was 2.6 times higher in its solution state with increasing water content of the poor solvent, indicating its typical AIE effect. In contrast, the TTFMN fluorescence intensity increased to 105 times the fluorescence intensity in solution with increasing water content of the poor solvent, indicating a more excellent AIE effect (as shown in fig. 2 and 3). The solid state emission peaks of both TFMN and TTFMN fall in the near infrared region (>650nm), indicating that they have deep red/near infrared fluorescence emission properties (as shown in figure 4).

The prepared compound with aggregation-induced emission property is distinguished from active oxygen generation and active oxygen type in solution by experiment

(1) Detection of total active oxygen

White light detection (22.1mW cm) using 2', 7' -dichlorodihydrofluorescein diacetate (DCFH-DA) as an ROS indicator^-2) ROS production in solution of AIE molecules under irradiation.

First, DCFH-DA in ethanol (1mM, 0.5mL) was added to 2mL NaOH solution (0.01M) and stirred at room temperature for 30 minutes to hydrolyze DCFH-DA to DCFH, which was then neutralized with 10mL 1 XPBS at pH 7.4 to give an activated ROS indicator (40. mu.M, 12.5mL), which was stored at 4 ℃ in the dark until needed. Then, the activated ROS indicator (40. mu.M) and AIE molecule were mixed in PBS to give final concentrations of ROS indicator and AIE molecule of 10. mu.M/5. mu.M, 50 nM/1. mu.M, respectively, and after white light irradiation for various periods of time, the fluorescence intensity of 2', 7' -dichlorofluorescein triggered by ROS production by AIE molecule was measured by fluorescence spectrometer (Edinburgh FS5) to reflect ROS production. The excitation wavelength is 488nm, and the fluorescence signal in the range of 490-600nm is collected.

(2)^·Detection of OH

Use of hydroxyphenyl fluorescein (HPF) as^·OH indicator, white light detection (22.1mW cm)^-2) In solution with AIE molecules under irradiation^·OH generation. The HPF mother liquor, AIE molecules and Crystal Violet (CV) mother liquor are mixed evenly in PBS to obtain test solutions with HPF and AIE molecules/CV final concentrations of 5 muM and 2 muM respectively. After different time of white light irradiation, usingFluorescence spectrometer (Edinburgh FS5) detects the fluorescence intensity of HPF, and the reaction^·OH generation. The excitation wavelength was 490nm and the fluorescence signal was collected in the range 500-620 nm.

(3)^·O₂ ^-Detection of (2)

Dihydrorhodamine (DHR123) is used as^·O₂ ^-Indicator, detecting white light (22.1mW cm)^-2) In solution with AIE molecules under irradiation^·O₂ ^-A situation arises. And uniformly mixing the DHR123 mother liquor and the AIE molecule mother liquor in PBS to obtain test solutions with DHR123 and AIE molecule final concentrations of 5 mu M and 2 mu M respectively. After white light irradiation for various times, DHR123 fluorescence intensity was measured by fluorescence spectrometer (Edinburgh FS5) and the reaction was performed^·O₂ ^-A situation arises. The excitation wavelength was 495nm and the fluorescence signals were collected in the range of 500-620 nm.

(4)¹O₂Detection of (2)

Using SOSG and ABDA as¹O₂Indicator, detecting white light (22.1mW cm)^-2) In solution with AIE molecules under irradiation¹O₂A situation arises. The SOSG stock solution and the AIE molecule/RB stock solution are mixed in PBS to obtain test solutions with final concentrations of SOSG and AIE molecule/RB of 5 muM and 2 muM respectively. After white light is irradiated for different time, the fluorescence intensity of the SOSG is detected by a fluorescence spectrometer, and the reaction is carried out¹O₂A situation arises. The excitation wavelength is 488nm, and the fluorescence signals in the range of 500-620nm are collected. As indicators for ABDA¹O₂And (3) detection, namely uniformly mixing the SOSG mother solution and the AIE molecule/RB mother solution in PBS to obtain test solutions with the final concentrations of the SOSG and the AIE molecule/RB being 20 mu M and 2 mu M respectively. After white light irradiation for different time, detecting ABDA absorption spectrum by using an ultraviolet absorption spectrometer, and recording absorption intensity reaction at the wavelength of 400nm¹O₂A situation arises.

The ability of TFMN and TTFMN to produce total ROS in PBS buffer was first investigated using DCFH-DA as an indicator. As shown in FIG. 5, DCFH alone was almost non-fluorescent, and with the addition of TFMN and TTFMN, DCFH fluorescence gradually increased with prolonged white light exposure, demonstrating thatBoth TFMN and TTFMN are effective in generating ROS under white light irradiation, and Δ E_S-TSmaller TTFMNs exhibit greater ROS production capability. Then, using HPF as^·OH indicator, investigation of TFMN and TTFMN production in PBS buffer^·The OH capacity. As shown in FIG. 6, both TFMN and TTFMN can be effectively generated under white light illumination^·OH, TTFMN are stronger^·OH generating ability. To further determine^·OH generation, DMPO was selected as a capture agent to test its ESR signal, and under the condition of TFMN and TTFMN existing respectively, after 1 minute of white light irradiation, obvious DMPO-^·The OH signal peaks and the TTFMN group showed stronger signals, further confirming that TTFMN is stronger^·OH generating capacity (as shown in FIG. 7). Dihydrorhodamine DHR123, SOSG and ABDA are selected as indicators to investigate the generation of TFMN and TTFMN^·O₂ ^-And¹O₂the results show that both TFMN and TTFMN can produce small amounts of^·O₂ ^-(as shown in FIG. 8), hardly any generation occurs¹O₂(as shown in fig. 9 and 10).

It can be seen that both TFMN and TTFMN can produce type I ROS, and TTFMN exhibits stronger type I ROS production efficiency.

Based on the same inventive concept, the embodiment of the invention also provides a preparation method of the nanoparticles of the compound with the aggregation-induced emission property.

Illustratively, 1mg TTFMN and 20mg amphipathic copolymer (85 mol% PEG-PLA and 15 mol%)^SATAT-PEG-PLA) in 1mL THF. Then, 1mL of the THF solution was added to 9mL of deionized water, followed by sonication with a probe sonicator for 2 minutes (45% output power). The mixture was then transferred to a dialysis bag (MWCO 3500Da) and dialyzed against deionized water for 24 hours. To completely remove THF, the water was changed every 4 hours. The resulting solution of TTFMN nanoparticles (TTFMN-NPs) is freeze-dried or concentrated by ultrafiltration before use. The Drug Loading (DLC) and the Encapsulation Efficiency (EE) of TTMFN were calculated according to the following formulas, respectively: DLC (wt%) × 100% (weight of TTMFN loaded/weight of TTFMN-NPs), EE (%) (weight of TTMFN loaded/weight of TTFM input)Weight of N). times.100%. Calculated using a previously determined standard absorption curve for TTFMN: DLC 4.4 wt%, EE 92.6%. TTFMN-NNP and TTFMN-PNPs were prepared in a manner similar to that described above, with 100 mol% PEG-PLA being the amphipathic copolymer matrix used to prepare TTFMN-NNP and 85 mol% PEG-PLA and 15 mol% TAT-PEG-PLA being the amphipathic copolymer matrix used to prepare TTFMN-PNPs.

Subsequently, the nanoparticles were characterized by a particle size analyzer (DLS) and Transmission Electron Microscopy (TEM). Among them, DLS results showed that TTFMN-NPs have water and particle sizes of about 70nm, favoring their EPR effect (as shown in FIG. 11); TEM results show that the non-hydrated particle size is between 30 and 60 nm. Absorption and emission peaks of TTFMN-NPs in aqueous solution were 501nm and 622nm, respectively (as shown in FIG. 13). The stability test results show that TTFMN-NPs have better stability in water, PBS buffer, 10% FBS in PBS (as shown in FIG. 12).

Further, the prepared nanoparticles as described above were tested for their nucleus targeting properties, illustratively,

(1) cell culture

The 4T1 cells are adherent cells, cultured in 1640 medium containing 10% Fetal Bovine Serum (FBS) at 37 deg.C and 5% CO₂The constant temperature incubator is used for culture.

(2) Endocytosis assay

4T1 cells were plated at 1X 10 per well⁵The density of individual cells was seeded into six-well plates and cultured for 24 hours. Then, the medium was replaced with fresh complete medium containing TTFMN-NNP, TTMFN-NP, TTMFN-NP (pretreated for 24 hours in PBS buffer at pH 5.0) or TTFMN-PNP and incubated at 37 ℃ for 3 hours. The concentration of the nano particles is 10 mu g mL^-1. Thereafter, the cells were gently washed 3 times with PBS, then trypsinized and the cells were rapidly harvested for flow analysis. The test results were analyzed using FlowJo10 software.

(3) Lysosomal escape and nuclear targeted delivery

4T1 cells were seeded at the appropriate density in a confocal dish and cultured for 24 hours. For lysosomal escape observation, cells were plated in cells containing TTFMN-NPs (2. mu.g mL)^-1) Fresh complete medium ofAfter 3 washes, the cells were incubated for 1h and 6h, respectively, and stained with LysoTracker Blue (LTB) for 30 minutes. Finally, the samples were washed with PBS and observed under CLSM. Conditions are as follows: excitation wavelength: LTB is 405nm, TTMFN-NP is 488 nm; an emission filter: LTB is 410-500nm, and TTMFN-NP is 550-750 nm.

To examine the effect of nuclear-nuclear targeted delivery, cells were incubated with TTFMN-NPs (2. mu.g mL)^-1) The cells were cultured in fresh complete medium for 1h, 6h and 12h, respectively, and after 3 washes, they were stained with Hoechst 33342 for 30 minutes. Finally, the samples were washed with PBS and imaged using CLSM. Conditions are as follows: excitation wavelength: hoechst 33342 is 405nm, TTMFN-NP is 488 nm; an emission filter: hoechst 33342 is 410-500nm, TTFMN-NP is 550-750 nm.

Due to the excellent cell membrane penetration of TAT, the endocytosis effect of TTFMN-NPs was first examined to evaluate the pH responsiveness of TTFMN-NPs. 4T1 cells were incubated with TTFMN-NNPs (encapsulation matrix 100 mol% PEG-PLA), TTFMN-NPs, TTFMN-NPs (pre-treated for 24 hours in PBS buffer at pH 5.0), and TTFMN-PNPs (encapsulation matrix 85 mol% PEG-PLA and 15 mol% TAT-PEG-PLA), respectively, for 3 hours. As shown in FIG. 14, the nanoparticle was significantly improved in the cell-entering effect after pretreatment for 24 hours at pH5.0 (MFI 10.8X 10) compared to TTFMN-NPs in which TAT was not activated³) And is obviously higher than TTFMN-NNPs of the negative control group and is equivalent to TTFMN-PNPs of the positive control group (MFI 11.8 multiplied by 10)³) Indicating that the SA-masked TAT can be successfully activated at pH 5.0. Subsequently, the distribution of TTFMN-NPs within 4T1 cells, acid triggered lysosomal escape capacity, was examined. As shown in fig. 15, TTFMN-NPs were mainly distributed to lysosomes after 1 hour incubation with 4T1 cells, indicating that TTFMN-NPs entered the cells via the endocytic pathway; with the prolonged incubation time of 6 hours, an obvious lysosome escape phenomenon appears, which indicates that the acidic environment of lysosome successfully activates TAT activity and triggers the lysosome escape. In addition, the nuclear targeting ability of TTFMN-NPs was further investigated by co-staining experiments with nuclear dyes. As shown in fig. 16, TTFMN-NPs were mainly distributed to lysosomes after 1 hour of incubation with 4T1 cells; prolonged incubation to 6 hours, some red fluorescence of TTFMN appeared thinThe perinuclear region; continuing to extend the incubation time to 12 hours, a significant amount of TTFMN-NPs entered 4T1 cells, transferred to the perinuclear region, attached to the nuclear membrane, and it was observed that a portion of the nanoparticles entered the interior of the nucleus. Taken together, the above results indicate that TTFMN-NPs can enter cells by endocytosis, TAT activity is activated in the acidic environment of lysosomes, and finally nuclear-targeted delivery of type I photosensitizers is achieved.

Further, the embodiment of the invention also verifies that the prepared compound with the aggregation-induced emission property can kill 4T1 cells in vitro by using photodynamic.

In an exemplary manner, the first and second electrodes are,

(1) intracellular reactive oxygen species detection

4T1 cells were seeded at the appropriate density in a confocal dish and cultured for 24 hours. First, cells were cultured in fresh medium (50. mu.gmL) containing TTFMN-NPs^-1) For 24 hours. After PBS wash, cells were incubated with fresh serum-free medium containing DCFH-DA (10. mu.M) for 20 minutes at 37 ℃. After washing, cells were exposed to 488nm laser irradiation (2% laser power) for 3 min before CLSM imaging. CLSM images were captured under 488nm excitation and fluorescence signals were collected over the range of 500 to 550 nm.

(2) Photodynamic-induced cytotoxicity assay

The MTT assay was used to assess the cytotoxicity of TTFMN-NPs in darkness and light. 4T1 cells were plated at 5X 10 per well³The density of individual cells was seeded in 96-well plates and incubated for 12 hours. The cells were then incubated with different concentrations of TTMFN-NPs in fresh medium. After 24 hours, the cells were exposed to white light (50mW cm)^-2) Followed by irradiation for 10 minutes. Meanwhile, the control group was treated in the dark. After further incubation for 24 hours, the medium was removed and washed 3 times with PBS. Cells were then incubated with fresh serum-free medium containing 10% MTT for 4 hours in the dark and aspirated, with 150uL DMSO per well. Finally, the absorbance at a wavelength of 570nm was measured by a microplate reader. Results are expressed as the percent survival of cells after different treatments relative to untreated control cells. Cell viability (%) ═ ODsample-ODbackground)/(ODcontrol-ODbackground)×100％。

(3) Photodynamic-induced apoptosis detection

4T1 cells were plated at 1X 10 per well⁵The density of individual cells was seeded in six-well plates and cultured for 24 hours. TTMFN-NPs (50. mu.g mL-1) were then added to the medium and incubated with the cells for 24 hours, followed by washing and replacement of fresh medium, white light (50mW cm)^-2) After 10 minutes of irradiation, the cells were incubated at 37 ℃ for a further 24 hours. Subsequently, the cells were digested, centrifuged (1000rpm, 5 min) and washed 3 times with PBS at 4 ℃. The instructions then stain the sample with the apoptosis kit Annexin V-APC and analyze by flow cytometry with excitation set at 633nm and emission filter at 640-680 nm.

Based on efficient nuclear delivery of the type I photosensitizer TTFMN, the effect of photodynamic killing of 4T1 cells was examined at the cellular level. First, examining the effect of generating ROS in TTFMN-NPs cells, as shown in FIG. 17, after 488nm laser irradiation, bright DCFH green fluorescence was detected in cells, while other control groups almost emitted no green fluorescence, indicating that TTFMN-NPs can generate ROS efficiently in cells. MTT experimental results show that TTFMN-NPs are low in dark toxicity, and good biocompatibility is shown; under light conditions, TTFMN-NPs showed concentration-dependent phototoxicity, indicating that TTFMN-NPs are capable of effectively photodynamically killing 4T1 cells (as shown in FIG. 18). The apoptosis flow analysis result shows that 75.4% of the cells in the TTFMN-NPs and the light treatment group are positive to APC, while other control groups have almost no positive rate (as shown in figure 19), which indicates that the TTFMN-NPs can effectively induce apoptosis through I type ROS mediated photodynamic under the light condition, and further cause cell death.

Further, the embodiment of the invention also verifies the photodynamic therapy mediated by in vivo near infrared fluorescence imaging of the mouse tumor.

In an exemplary manner, the first and second electrodes are,

(1) animal and tumor model establishment

Four-week-old male BALB/c nude mice were purchased from Experimental animals technology, Inc., Viton, Beijing. All animals were acclimatized to animal feeding prior to the experimentThe environment is one week, and the breeding is carried out under the condition without pathogens. To establish a 4T1 tumor-bearing mouse model, 4T1 breast cancer cells (5X 10) suspended in 100. mu.L PBS buffer⁵) Injected subcutaneously to the ventral side of each mouse. After about 10 days, a tumor volume of about 100mm can be obtained³The tumor-bearing mouse of (1).

(2) Mouse tumor in vivo near infrared fluorescence imaging

In vivo near infrared fluorescence imaging of mouse tumors was performed on a commercial IVIS Spectrum imaging system (PerkinElmer). Tumor-bearing mice were injected with TTMFN-NPs (10mg TTFMN kg) via tail vein^-1). Fluorescence imaging was performed at different times (0, 1, 3, 6, 12 and 24 hours) after injection. To assess the tissue distribution of TTMFN-NPs, mice will be sacrificed 24 hours after injection. Major organs (heart, liver, spleen, lung and kidney) and tumors were collected and the organ surfaces were then washed with saline for near infrared fluorescence imaging and quantitative analysis.

(3) Photodynamic therapy of mouse tumors

When the inoculated tumor grows to about 100mm³Tumor-bearing mice were randomized into four groups (n-5) and received saline, saline + light, TTMFN-NPs and TTFMN-NPs + light, respectively. For the "saline" and "TTFMN-NPs" groups, 200. mu.L of physiological saline and TTFMN-NPs (10mg TTFMN kg), respectively^-1) Intravenous injection into 4T1 tumor-bearing mice was performed without subsequent white light illumination. For the "saline + Lighting" and "TTFMN-NPs + Lighting" groups, white light irradiation (100mW cm) was performed at the tumor sites of mice in each group after 12 hours of intravenous injection of saline and TTMFN-NPs^-2) For 20 minutes. Treatment was performed every three days for 15 days, and mouse body weight and tumor volume were recorded every 3 days over 15 days to find changes in body weight and relative tumor volume. The tumors were measured with a vernier caliper and according to the formula V ═ a × b²The tumor volume was calculated 2. (a: length of tumor; b: width of tumor). The relative tumor volume was calculated as RTV-V/V₀(V₀Initial tumor volume).

Based on the excellent photodynamic killing effect of TTFMN-NPs on the cell level, the TTFMN-NPs are used for the photodynamic therapy guided by fluorescence imaging in a mouse tumor model. First, the ability of TTFMN-NPs to be used for in vivo tumor imaging in mice was evaluated, and 4T1 tumor-bearing mice were subjected to in vivo animal imaging after different times of tail vein injection of TTFMN-NPs, respectively (as shown in FIG. 20). Due to the appropriate size and potent EPR effect of TTFMN-NPs, red fluorescence of TTFMN was observed in the tumor region 1 hour after tail vein injection, indicating rapid enrichment of TTFMN-NPs. The enrichment of TTFMN-NPs in the tumor area is gradually increased with time, and the fluorescence intensity reaches the maximum after 12 hours of injection, which indicates that the enrichment of TTFMN-NPs in the tumor area reaches the maximum. The imaging has extremely high contrast thanks to its typical AIE properties, high quantum yield in the aggregated state and near infrared emission. Subsequently, the fluorescence of the tumor area gradually decreased due to the physiological metabolism of the mouse. Even so, after 24 hours of tail vein injection, fluorescence signals can still be detected at the tumor site, which indicates that TTFMN-NPs have long retention effect at the tumor site and are beneficial to photodynamic therapy.

Based on the fact that the enrichment amount of TTFMN-NPs in tumors reaches the maximum 12 hours after injection, white light irradiation is carried out on the tumors of mice for 20 minutes after 12 hours of tail vein injection of TTFMN-NPs, and the mice are used as an experimental group. Meanwhile, the normal saline group, the single light group or the single TTFMN-NPs group served as a control group. As shown in fig. 21, the tumor volume of the control group was greatly increased and the growth rate of the tumor volume of the TTFMN-NPs + light group was significantly inhibited with time, indicating that the TTFMN-NPs could effectively kill cancer cells with photodynamic action, effectively inhibit tumor growth and reduce tumor volume under light condition. After the treatment period, the tumors of mice in TTFMN-NPs + light group were significantly smaller than those in other control groups, and showed higher tumor inhibition rate (as shown in FIG. 21). Mouse tumor sections were histologically and immunohistochemically stained for relevant anti-tumor mechanisms (as shown in figure 22). In conjunction with fig. 23, the H & E staining results show that after photodynamic treatment, tumor tissue showed significant vacuolization, substantial nuclear confirmation and significant nuclear condensation, indicating significant damage. TUNEL staining results showed that TTFMN-NPs + light group showed more apoptotic signal (red fluorescence) than other control group. Similarly, the TTFMN-NPs + light group showed less Ki67 positive cell proliferation signal (green fluorescence) and CD31 positive neovascular signal (green fluorescence). The results show that the cell nucleus targeted I-type photodynamic therapy can effectively induce apoptosis, inhibit tumor proliferation and angiogenesis by generating active oxygen in situ in the cell nucleus, thereby inhibiting the growth of the tumor.

It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.

Claims (10)

1. The compound with aggregation-induced emission property is characterized by having the following molecular structural formula

Wherein R is¹、R²Each independently selected from H,

One kind of (1).

2. A process for preparing a compound of claim 1, wherein when R is¹、R²When both are H, the method comprises the steps of:

providing a compound I;

dissolving the compound I and malononitrile into ethanol, and heating and refluxing to obtain the R¹、R²Compounds that are all H;

wherein the molecular structural formula of the compound I is

3. The process for the preparation of a compound according to claim 2, wherein the process for the preparation of compound I comprises the steps of:

adding 4-bromotriphenylamine, 5-formaldehyde furan-2-boric acid, [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride and potassium carbonate into a reaction vessel;

and adding a mixed solvent into the reaction vessel, and heating the reaction vessel under an inert atmosphere to obtain the compound I.

4. The method for producing a compound according to claim 3, wherein the mixed solvent contains methanol and toluene.

5. A process for preparing a compound of claim 1, wherein when R is¹Is H, R²Is composed of

The method comprises the following steps:

providing a compound IV;

dissolving the compound IV and malononitrile in dry ethanol, and heating and refluxing to obtain R¹Is H, R²Is composed of

A compound of (1);

wherein the molecular structural formula of the compound IV is

6. The method of claim 5, wherein the step of preparing compound IV comprises the steps of:

providing a compound III;

adding the compound III, 5-formaldehyde furan-2-boric acid, [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride and potassium carbonate into a reaction vessel;

adding a mixed solvent containing methanol and toluene into the reaction container, and heating the reaction container under an inert atmosphere to obtain a compound III;

the molecular structural formula of the III is

7. The method of claim 6, wherein the preparation of compound III comprises the steps of:

providing a compound II;

adding the compound II, p-bromoiodobenzene, 1, 10-phenanthroline, cuprous iodide and potassium hydroxide into a reaction vessel;

adding toluene under the protection of inert gas, and heating the reaction container to obtain the compound III;

the molecular structural formula of the compound II is

8. The method of claim 7, wherein the step of preparing compound II comprises the steps of:

dissolving 1- (4-bromophenyl) -1, 2, 2-triphenylethylene, aniline, tri-tert-butylphosphine, tris (dibenzylideneacetone) dipalladium and sodium tert-butoxide in toluene;

and (3) carrying out reaction under the protection of inert gas and heating condition to obtain the compound II.

9. The method for preparing a compound according to claim 8, wherein the heating conditions are a heating temperature of 110 ℃ to 130 ℃.

10. Use of a compound with aggregation-induced emission properties according to claim 1 for nuclear-targeted imaging or for nuclear-targeted photodynamic therapy.

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